Srf(-/-) ES cells display non-cell-autonomous impairment in mesodermal differentiation.

The serum response factor (SRF) transcription factor is essential for murine embryogenesis. SRF+(-/-) embryos stop developing at the onset of gastrulation, lacking detectable mesoderm. This developmental defect may reflect cell-autonomous impairment of SRF(-/-) embryonic cells in mesoderm formation. Alternatively, it may be caused by a non-cell-autonomous defect superimposed upon inappropriate provision of mesoderm-inducing signals to primitive ectodermal cells. We demonstrate that the ability of SRF(-/-) embryonic stem (ES) cells to differentiate in vitro into mesodermal cells is indeed impaired. However, this impairment can be modulated by external, cell-independent factors. Retinoic acid, but not dimethylsulfoxide, permitted activation of the mesodermal marker gene T(Bra), which was also activated when SRF was expressed in SRF(-/-) ES cells. Embryoid bodies from SRF(-/-) ES cell aggregates also activated mesodermal marker genes, but displayed unusual morphologies and impairment in cavitation. Finally, in nude mice, Srf(-/-) ES cells readily differentiated into mesodermal cells of SRF(-/-) genotype, including cartilage, bone or muscle cells. We demonstrate that SRF contributes to mesodermal gene expression of ES cells and that SRF(-/-) ES cells display a non-cell-autonomous defect in differentiation towards mesoderm.

Serum response factor is essential for mesoderm formation during mouse embryogenesis.

The transcription factor serum response factor (SRF), a phylogenetically conserved nuclear protein, mediates the rapid transcriptional response to extracellular stimuli, e.g. growth and differentiation signals. DNA- protein complexes containing SRF or its homologues function as nuclear targets of the Ras/MAPK signalling network, thereby directing gene activities associated with processes as diverse as pheromone signalling, cell-cycle progression (transitions G0-G1 and G2-M), neuronal synaptic transmission and muscle cell differentiation. So far, the activity of mammalian SRF has been studied exclusively in cultured cells. To study SRF function in a multicellular organism we generated an Srf null allele in mice. SRF-deficient embryos (Srf -/-) have a severe gastrulation defect and do not develop to term. They consist of misfolded ectodermal and endodermal cell layers, do not form a primitive streak or any detectable mesodermal cells and fail to express the developmental marker genes Bra (T), Bmp-2/4 and Shh. Activation of the SRF-regulated immediate early genes Egr-1 and c-fos, as well as the alpha-Actin gene, is severely impaired. Our study identifies SRF as a new and essential regulator of mammalian mesoderm formation. We therefore suggest that in mammals Ras/MAPK signalling contributes to mesoderm induction, as is the case in amphibia.

[Elimination of introns from the interleukin 1 beta genome by DNA amplification and expression of interleukin 1 beta in Escherichia coli]. / Udalenie intronov metodom amplifikatsii DNK iz genomnogo gena interleikina 1 beta i ékspressiia gena interleikina 1 beta v Escherichia coli]

The use of the polymerase chain reaction was proposed for intron excision from genomic genes with known nucleotide sequences. Three exons (5, 6 and 7) of genomic interleukin 1 beta gene were amplified by means of thermostable DNA polymerase TthI from Thermus thermophilus on the base of cloned in M13 phage human genomic interleukin 1 beta gene. Synthetic oligonucleotides complementary to sequences flanking exons were used as primers. The fragments obtained by exon DNA amplification were joined in the correct order due to reciprocal complementation of end sequences, that was foreseen during synthesis of oligonucleotide primers followed by amplification of the enlarged fragments. As a result the structural interleukin-1 beta gene consisting of three exons was assembled. DNA sequences carrying the ATG initiation codon and XbaI recognition site at the 5'-end, and PstI recognition site at the 3'-end (essential for insertion into the expression vector) were formed by the additional end sequences of primers. The nucleotide sequence analysis of the obtained structural gene revealed its complete identity with natural interleukin 1 beta human gene. We created the expression vector pPR114 with phage lambda promoter PR thermo-inducible in case of the cIts857 repressor presence in cells. It was used for expression of the present gene. The interleukin 1 beta synthesized in E. coli had biological activity.

[An experimental study of human recombinant gamma interferon with a proteolytically reduced C-end area of the protein]. / Eksperimental'noe izuchenie rekombinantnogo chelovecheskogo gamma-interferona s proteoliticheski redutsirovannym C-kontsevym uchastkom belka.

The work presents the results of experimental study of gamma interferon obtained by gene engineering techniques on the basis of Escherichia coli producer strains. The study has revealed that gamma interferon, whose molecular weight is 15 KD, due to intracellular proteolytic degradation shows the absence of some amino acids at the C-end of protein and is electrophoretically homogeneous, while its antiviral, antiproliferative and immunomodulating effects are less pronounced than those of gamma interferon with a molecular weight of 18 KD.

Drosophila homolog of the murine Int-1 protooncogene.

We have isolated phage clones from Drosophila melanogaster genomic and cDNA libraries containing a sequence homologous to the murine Int-1 protooncogene. The Drosophila gene is represented by a single locus at position 28A1-2 on chromosome 2. The gene is expressed as a 2.9-kilobase-long polyadenylylated mRNA in embryo, larval, and pupal stages. It is hardly detectable in adult flies. The longest open reading frame of the cDNA clone corresponds to a protein 469 amino acids long. Alignment of the predicted amino acid sequences shows that the Drosophila protein is 86 amino acids longer than its murine counterpart. In spite of the difference in length, the two proteins are highly conserved with an overall sequence homology of 54%. Both Drosophila and murine Int-1 proteins begin with a hydrophobic leader sequence and contain cysteine residues and sites for glycosylation (four in the murine protein and one in the Drosophila protein) in conserved positions, suggesting that they play important functional roles.

[Bank of sequences specific for human genome. I. The structure of one of the repeats in the Alu family]. / Poluchenie banka posledovatel'nostei, spetsifichnykh dlia genoma cheloveka. I. Struktura odnogo iz povtorov Alu-semeistva.

A bank enriched in sequences specific for the human genome was obtained. In course of the analysis, a clone containing an Alu family repeat was identified and its primary structure determined.

[Primary structure of the alpha subunit of Na+,K+-ATPase. II. Isolation, reverse transcription and cloning of messenger RNA]. / Pervichnaia struktura alpha-sub''edinitsy Na+,K+-ATP-azy. II. Vydelenie, obratnaia transkriptsiia i klonirovanie matrichnoi RNK.

Messenger RNA, coding for the alpha-subunit of the Na+, K+-ATPase, was isolated from outer medulla of pig kidney. Within 25S-26S region the mRNA yields a band of specific hybridization with three oligonucleotide probes synthesized according to data on structures of three peptides isolated from the tryptic hydrolysate of the protein. Translation of the enriched poly(A+)-fraction of RNA in Xenopus laevis oocytes followed by the immunochemical identification of the products confirmed the presence of RNA coding for the desired protein. This RNA preparation was used for synthesis and cloning of double stranded cDNA.

[Problems associated with the use of highly degenerated oligonucleotide probes. Identification of mRNA and cDNA clones corresponding to the gene for the alpha subunit of Na+,K+-ATPase]. / Problemy, sviazannye s ispol'zovaniem oligonukleotidnykh zondov s bol'shoi stepen'iu vyrozhdennosti. Identifikatsiia mRNK i klonov kDNK, sootvetstvuiushchikh genu alpha-sub''edinitsy Na+,K+-ATP-azy.

Oligonucleotides deduced from the amino acid sequence of a hexapeptide Lys-Asp-Phe-Ala-Glu-Asn were synthesized and used as probes to screen a pig kidney cDNA library for a specific DNA sequence coding for the alpha-subunit of Na+, K+-ATPase. It was shown that the mixed oligoprobe, consisting of 64 heptadecamers, could be only suitable for mRNA blot analysis. To identify the clones with specific cDNA inserts, mixed oligoprobes were fractionated by HPLC technique. For the same purpose a new set of oligonucleotides, synthesized as four groups of 16 different heptadecamers each, was used.

[Enzymatic synthesis and properties of DNA, complementary to foot and mouth disease virus RNA]. / Fermentativnyi sintez i svoistva DNK, komplementarnoi RNK virusa iashchura.

The RNAs of the aphthosa virus, serotypes A and O were isolated and characterized. The complementary DNAs used in experiments on molecular hybridization with matrix RNAs were synthesized on virion RNAs by means of reverse transcriptase.

[Simple method for DNA fraction enrichment with unique genes]. / Prostoi sposob obog a shcheniia praktsii DNK po soderzhaniiu unikal'nykh genov.

The conditions for RNA--DNA molecular hybridization in 15% phenolic suspension, allowing to extend the temperature range of the R-loop formation and render the method insensitive to the nucleotide composition of RNA are proposed. The method described results in a 100-fold enrichment of the globin-gene content in a few days. The method was used for enrichment of rat DNA preparations with globin genes.